susceptibility testing issues (web)
DESCRIPTION
Antimicrobial susceptibility testingSome (unresolved) issuesAims of this talkcomparing different susceptibility test methods do different test results matter? specific testing issuesTesting methodsDisc Broth dilution Automated methods EtestTesting methodsDisc Broth dilution Automated methods Etest Inoculum Disc strength Media type pH Anionic content ReproducibilityTesting methodsDisc = Broth dilution = Automated methods = Etest Essential agreement +/- 1 dilution of the acceptedTRANSCRIPT
Antimicrobial susceptibility testing
Some (unresolved) issues
Aims of this talkAims of this talk
comparing different susceptibility test
methods
do different test results matter?
specific testing issues
Testing methodsTesting methods
Disc
Broth dilution
Automated methods
Etest
Testing methodsTesting methods
Disc
Broth dilution
Automated methods
Etest
Inoculum
Disc strength
Media type
pH
Anionic content
Reproducibility
Testing methodsTesting methods
Disc
=
Broth dilution
=
Automated methods
=
Etest
Essential agreement
+/- 1 dilution of the
accepted “gold standard”
Testing methodsTesting methods
Essential
agreement
4
2
1
0.5
0.25
MIC (mg/L)
1
Essential agreementEssential agreement
Are these two methods in essential agreement?
MIC testing by Method 1 & Method 2
0
1
2
3
4
5
6
7
8
0.25 0.5 1 2 4 8 16 32
MIC values
no of isolates w
ith MIC
MIC testing by Method 1
0
1
2
3
4
5
6
7
8
0.25 0.5 1 2 4 8 16 32
MIC values
no of isolates w
ith MIC
Categorical agreementCategorical agreement
Method 1 has a lot more “S” than Method 2
– Even though both methods have excellent
essential agreement
Susceptible ≤ 2; Resistant > 2
MIC distributionsMIC distributions
breakpoint
no. of resistant strains
no. of susceptible strains
No
of
stra
ins
MIC
MIC distributionsMIC distributions
breakpoint
no. of resistant strains
(with the second method)
no. of susceptible strains
(with the 2nd method)
+ 1 log dilution
MIC distributionsMIC distributions
breakpoint
no. of resistant
strains
no. of susceptible strains
MIC distributionsMIC distributions
breakpoint
no. of resistant
Strains
(with 2nd method)
no. of susceptible strains
(with 2nd method)
+ 1 log dilution
Categorical errorsCategorical errors
Intermediate
error
Intermediate
error
False
resistance
False
susceptibility
Interpretation
Minor errorIS or R
Minor errorS or RI
Major errorSR
Very major
error
RS
Error
category
“Gold
standard”
Test
system
Susceptibility testing methodsSusceptibility testing methods
Multiple factors may affect susceptibility testing
results
Different testing methods need to agree:
– Essential agreement
– Categorical agreement
Automated systemsAutomated systems
Guidance for Industry and FDA
Class II Special Controls Guidance Document: Antimicrobial Susceptibility Test (AST) Systems
Document issued on: August 28, 2009
EN ISO 20776-1:2006
Clinical laboratory testing and in vitro diagnostic test systems - Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices -Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases (ISO 20776-1:2006)
AST systems AST systems -- FDAFDA
% categorical and essential agreement
> 89.9%.
major error rate
< 3%
very major error rate (based on the number of resistant organisms tested)
1.5% - 7.5%
AST systems AST systems -- CLSICLSI
% categorical and essential agreement
major error rate
very major error rate (based on the number of resistant organisms tested)
GramGram--positivepositive
agentsagents
DaptomycinDaptomycin
Cyclic lipopeptide antibiotic
Bactericidal
Bacteraemia and skin/soft tissue infections
Acts on cell wall
membrance
Calcium dependant
action
DAPTOMYCIN
DaptomycinDaptomycin
MIC’s decrease with increasing Ca2+
concentration
Etest:
calcium supplemented strips
Microbroth dilution:
calcium supplemented broth
DAPTOMYCIN
DaptomycinDaptomycin
Etest v.s. microbroth dilution
Etest MIC higher by 0.5 – 1 dilution
Lot –related elevation in QC?
DAPTOMYCIN
Sader HS, et al. Nine-hospital study comparing broth microdilution and Etest method results for vancomycin and daptomycin against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2009 Jul;53(7):3162-5.Friedrich L, et al. Evidence for daptomycin Etest lot-related MIC elevations for Staphylococcus aureus. Diagn Microbiol Infect Dis. 2009 Nov;65(3):306-11.
VANCOMYCIN
VancomycinVancomycin
Glycopeptide
Bactericidal (bacteriostatic?)
Diffuses slowly into agar
MIC testing (broth, agar), Etest and Vitek
methods
(no disc)
VancomycinVancomycin
Etest v.s. microbroth dilution
Etest MIC higher by 1 dilutionVANCOMYCIN
Sader HS, et al. Nine-hospital study comparing broth microdilution and Etest method results for vancomycin and daptomycin against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2009 Jul;53(7):3162-5.
VancomycinVancomycin
Etest v.s. microbroth dilution v.s. Vitek
Vitek < broth microdilution < Etest
VANCOMYCIN LOW HIGH
Phoenix,Microscan
Agar dilution,Sensititre
Jana M. Swenson, et al. Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus. J Clin Microbiol. 2009 July; 47(7): 2013–2017.
-1 +10
doubling dilution
VancomycinVancomycin
Etest v.s. microbroth dilution v.s. Vitek
Vitek
VANCOMYCIN LOW HIGH
Agar dilution,Sensititre
Jana M. Swenson, et al. Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus. J Clin Microbiol. 2009 July; 47(7): 2013–2017.
-1 +10
doubling dilution
VancomycinVancomycin
Etest v.s. microbroth dilution v.s. Vitek
Etest
VANCOMYCIN LOW HIGH
Phoenix,Microscan
Jana M. Swenson, et al. Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus. J Clin Microbiol. 2009 July; 47(7): 2013–2017.
-1 +10
doubling dilution
VancomycinVancomycin
Agar screen plate?
BHI agar with 3 mg/L vancomycin
100% sensitivity, 65% specificity
VANCOMYCIN
Carey-Ann D. et al. Novel Screening Agar for Detection of Vancomycin-NonsusceptibleStaphylococcus aureus. J Clin Microbiol. 2010 March; 48(3): 949–951.
Ability to detect true positives
Ability to detect true negatives
TigecyclineTigecycline
Glycycline
(related structure to tetracyclines)
Liable to oxidation
Available for disc (FDA), broth dilution, Etest and Vitek
TIGECYCLINE
Tigecycline & EtestTigecycline & Etest
broth microdilution < EtestTIGECYCLINE
0 1-1doubling dilution
FOR:
Acinetobacter spp., S. pneumoniae, Serratia marcescens
Pillar CM, et al. In vitro activity of tigecycline against gram-positive and gram-negative pathogens as evaluated by broth microdilution and Etest.J Clin Microbiol. 2008 Sep;46(9):2862-7. Epub 2008 Jul 2.
Tigecycline & broth dilutionTigecycline & broth dilution
Needs fresh Mueller-Hinton broth
– MIC’s in aged broth increased by up to 1 dilution
– caused by oxidative effect of dissolved oxygen
May also be a problem for commercial
microbroth kits (e.g. Sensititre)
For agar dilution, use freshly steamed agar
TIGECYCLINE
Patricia A. Bradford, et al. Tigecycline MIC Testing by Broth Dilution Requires Use of Fresh Medium or Addition of the Biocatalytic Oxygen-Reducing Reagent Oxyrase To Standardize the Test Method. Antimicrob Agents Chemother. 2005 September; 49(9): 3903–3909.
Hope R, et al. Effect of medium type, age and aeration on the MICs of tigecycline and classical tetracyclines. J Antimicrob Chemother. 2005 Dec;56(6):1042-6.
Tigecycline & agar methodsTigecycline & agar methods
Zone diameters may change depending on
brand of agar used
– affects Acinetobacter spp., Enterobacteriaceae
Applies to disc and Etest
TIGECYCLINE
Fernández-Mazarrasa C, et al. High concentrations of manganese in Mueller-Hinton agar increase MICs of tigecycline determined by Etest. J ClinMicrobiol. 2009 Mar;47(3):827-9.
Canigia LF, Bantar C. Susceptibility testing of tigecycline against Acinetobacter spp. by disc diffusion method: withdrawing a therapeutic option by varying the Mueller-Hinton agar? J Antimicrob Chemother. 2008 Dec;62(6):1463-4.
GramGram--negativenegative
agentagent
Polymyxin B & agar methodsPolymyxin B & agar methods
Difficult antibiotic to test:
narrow zone diameters
affected by cations (especially P. aeruginosa)
Etest values affected by brand of media & different lots of media
Disc testing is not reliable
POLYMYXIN
B
Lo-Ten-Foe JR, et al.Comparative evaluation of the VITEK 2, disk diffusion, etest, broth microdilution, and agar dilution susceptibility testing methods for colistin in clinical isolates, including heteroresistant Enterobacter cloacae and Acinetobacter baumannii strains. Antimicrob Agents Chemother. 2007 Oct;51(10):3726-30.
Polymyxin B & agar methodsPolymyxin B & agar methods
Heterogenous resistance: Enterobacter spp.,
Stenotrophomonas maltophiliaPOLYMYXIN
B
Polymyxin B & other methodsPolymyxin B & other methods
Vitek testing:
caution required: may not detect heterogenous
resistance
MIC’s tend to be lower
Broth dilution:
“Gold standard” method
POLYMYXIN
B
Tan TY, Ng SY. Comparison of Etest, Vitek and agar dilution for susceptibility testing of colistin. Clin Microbiol Infect. 2007 May;13(5):541-4. Epub 2007 Mar 19.
SummarySummary
How to evaluate results from different testing
methods
Problematic antibiotics – importance of quality
assurance
Know your bacterial population and the
meaning of different testing methods